PROJECT SUMMARY/ABSTRACT Lassa virus (LASV), the causative agent of Lassa fever (LF), is the arenavirus with the highest impact in public health. Because the large LASV endemic regions in West Africa and size of the population at risk of infection, LF is arguable one of the viral hemorrhagic fevers with the highest burden in public health. Epidemiological studies support the implementation of a live-attenuated vaccine (LAV) as the most feasible approach to control LF. Advances in biotechnology and molecular virology have provided new opportunities for rational design of LAV with optimal balance of safety and efficacy. Our lead vaccine candidate, reassortant ML29/Jos, is currently the only LASV vaccine candidate capable to induce sterilizing cell-mediated immunity, cross- protection against genetically distantly related LASV, and to prevent death in post-exposure application. Moreover, ML29/Jos was genetically stable and well tolerated in all animal models of LASV infection tested, including SIV-immune compromised rhesus macaques. However, lack of FDA-compliant documentation to track the generation and passage history of ML29/Jos poses a major roadblock for its clinical development. Our overall working hypothesis is that the use of a genetically re-designed form of ML29/Jos generated via reverse genetics from cloned cDNAs (rML29/Jos) will provide us with a powerful vaccine platform to develop a LASV LAV that is genetically well-defined, stable and with a well-documented passage history, as well as improved safety and broad cross-protective capabilities. To test this hypothesis we propose: 1) To characterize genetically and phenotypically our recently rescued rML29/Jos to confirm its properties of LAV. 2) To assess the contributions of GPC and NP specific mutations to rML29/Jos attenuation. 3) To determine the safety, immunogenicity and cross-protective efficacy of rML29/Jos in experimental rodents. Cross-reactivity and safety features of rML29 will be improved, if need it, by generating tri-segmented forms of rML29/Jos expressing antigens from genetically distantly related LASV lineages and by implementing S gene rearrangements known to promote arenavirus attenuation, respectively. 4) To determine the safety, immunogenicity and protective efficacy of rML29-based LAV lead candidate in a cynomolgus monkey model of LASV infection.